The complete chloroplast genome of Primula vialii (Primulaceae), an ornamental plant

Abstract Primula vialii Delavay ex Franch. (1905) is an alpine species with an ornamental value. In this study, we sequenced, assembled, and annotated the chloroplast genome of P. vialii. The results showed that it was a double-stranded, closed circular DNA with 154,897 bp in length, comprising a small single-copy (SSC) region of 17,766 bp, a large single-copy (LSC) region of 85,379 bp and a pair of inverted repeat (IR) regions of 25,876 bp. A total of 113 unique genes were annotated, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The phylogenetic analysis revealed that P. vialii is closely related to Primula flaccida. The cp genomic data will be useful for systematics and evolutionary studies of Primula.


Introduction
Primula vialii Delavay ex Franch. (1905) is a perennial herb in the genus Primula (Primulaceae), which widely distributed in wet meadows and valleys at an altitude of 2800-4000 m in Northwest Yunnan and Southwest Sichuan of China (Hu and Kelso 1996). With its blue and red spires of flowers (Figure 1 CONTACT Yuan Huang huangyuanynnu@aliyun.com School of Life Sciences, Yunnan Normal University, Kunming, P. R. China Supplemental data for this article can be accessed online at https://doi.org/10. 1080/23802359.2023.2202268. this extraordinary species presents an unique feature in Primula (Richards 2003). Because of its dark green basal leaves, it is invariably used as an ornamental foliage plant during vegetative growth period. However, previous studies have focused on tissue culture and rapid propagation (Li et al. 2005) of P. vialii rather than systematics and evolutionary studies. Here, we have sequenced and analyzed the chloroplast (cp hereafter) genome of P. vialii, which contributes to provide more cp genome genetic information further evolutionary research and phylogenetic study of Primula.

Materials and methods
Fresh leaves of an individual P. vialii were collected from the Alpine Botanical Garden of Lijiang City, Yunnan Province, China (26.997 N,100.201 E). The voucher specimen  was deposited at the Herbarium of Yunnan Normal University (Kunming, China; Jianlin Hang, hjlyuun@ 163.com). Total genomic DNA was extracted from the fresh leaves using the modified CTAB method (Porebski et al. 1997) and stored in Huang's Lab of Yunnan Normal University. The total DNA was fragmented into 300 bp short Figure 2. Genomic map of Primula vialii chloroplast genome generated by CPGview (http://www.1kmpg.cn/cpgview/). The species name is shown in the left top corner. The map contains six tracks in default. From the center outward, the first track shows the dispersed repeats. The dispersed repeats consist of direct and Palindromic repeats, connected with red and green arcs. The second track shows the long tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars with different colors. The small single-copy (SSC), inverted repeat (IRA and IRB), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. The base frequency at each site along the genome will be shown between the fourth and fifth tracks. The genes are shown on the sixth track. The optional codon usage bias is displayed in the parenthesis after the gene name. Genes belonging to different functional groups are color-coded. Genes drawn inside the circle are transcribed clockwise, and those outside are transcribed counterclockwise.
sequences following the manufacture's protocoal (Illumina Inc., USA) to construct a DNA library, then sequenced on the Illumina Hiseq X Ten sequencing platform. We obtained 18,438,252 filtered reads of paired-end sequences (2 Â 150 bp) and assembled the complete cp genome using NOVOPlasty v4.3.1 (Dierckxsens et al. 2017). To verify the accuracy of the assembly, we further mapped our clean reads back to the assembled cp genome to assess the depth of coverage ( Figure S1). Taking the cp genome of Primula flaccida (Genbank accession No. NC_053595) as the reference sequence, the assembled genome was annotated using Geneious v2020.1.1 software (Kearse et al. 2012). The annotated cp genome sequences of P. vialii were deposited in the GenBank database under accession No. ON584545. The genome map and cis/trans-splicing genes map ( Figure S2) was drawn using the CPGView program (http://www.1kmpg.cn/ cpgview).
In order to investigate the phylogenetic relationship of P. vialii in the genus Primula, we downloaded 39 Primula cp genomes and 5 Primulaceae cp genomes (Glaux maritima (Liu et al. 2021), two Lysimachia species and two Androsace species) as outgroups from NCBI. The 45 cp genome sequences were aligned by software MAFFT v7.47 (Katoh and Standley 2013), then constructed a maximum likelihood tree using the software IQ-TREE 2 (Katoh and Standley 2013;Minh et al. 2020) under TVM þ F þ R3 best-fit model according to Bayesian information criterion (Kalyaanamoorthy et al. 2017). Branch supports were tested using ultrafast bootstrap (UFBoot) (Hoanget al. 2018) and SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010) with 10,000 replicates.

Results
The cp genome of P. vialii was assembled as a doublestranded, closed-circular DNA molecule of 154,897 bp in length (Figure 2), with an average coverage of 864.9 and GC contents of 36.9%. It has a typical quadripartite structure and comprises a large single-copy (LSC) region of 85,379 bp and a small single-copy (SSC) region of 17,766 bp, separated by two inverted repeat (IR) regions of 25,876 bp each. The GC contents of these four regions are 34.8%, 30.3%, 42.6%, 42.6%, respectively. In total, the cp genome has 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, of which 113 genes, 79 protein-coding genes, 30 tRNA genes, 4 rRNA genes are unique, respectively. Most of these genes are single-copy genes, while 8 protein-coding genes, 7 tRNA genes, and 4 rRNA genes were duplicated in the IR regions. The phylogentic tree (Figure 3) indicated the relationship between Primula, Androsace, Lysimachia, and Glaux in Primulaceae, and 40 Primula species formed a monophyletic clade. In addition, P. vialii is closely related to P. flaccida and P. cawdoriana in Sect. Soldanelloides.

Discussion and conclusion
Within Primula clade, P. vialii, P. flaccida, and P. cawdoriana formed a monophyletic clade and were sister to each other (Figure 3), which is consistent to previous studies based on noncoding cp DNA sequence (Mast et al. 2001). Mast (2001) concluded that P. vialii belongs to Sect. Muscarioides, and members of Sect. Muscarioides are interdigitated with members of Sect. Soldanelloides. Although we reported the first complete cp genome of Sect. Muscarioides in this study, other complete cp genomes of Sect. Muscarioides are not sequenced yet. Therefore more phylogenetic researches are needed to exactly confirm the relationship of Sect. Muscarioides and Sect. Soldanelloides. Overall, the complete cp genome of P. vialii can be subsequently used for phylogenetic, taxonomic, and evolutionary studies of Sect.

Ethics statement
This study does not involve ethical issues. The sample of this study is not a protected plant, and the sample collection has been approved by Lijiang Alpine Botanical Garden.

Authors contributions
Yunqi Liu performed the design of experiments, writing the original draft, and data curation. Shubao Wang, Li Zhang, and Rui Li performed the collection of P. vialii form wild fields, species identification, DNA extraction, and data analysis. Yuan Huang prepared the resources, supervised the project, made revisions to the manuscript, and final approval of the version to be published.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at [https://www.ncbi.nlm.nih.gov/] under accession no. ON584545. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA841839, SRR19391427, and SAMN28626130, respectively.